Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus responsible for recent major outbreaks of polyarthralgia in countries of the Indian Ocean region. This important human pathogen causes an acute febrile illness characterized by a maculopapular rash and incapacitating arthralgia, which can evolve to chronic arthritis. CHIKV, like other alphaviruses, is transmitted through the bite of an infected mosquito. The virus is currently endemic in Africa, India, and Southeast Asia, but it has recently spread to new areas with attack rates as high as 75 percent. Due to the high prevalence of the CHIKV insect vectors in many parts of the world, the potential for CHIKV to cause significant epidemics in uninfected regions remains high. Little is known about how this virus interacts with the host and the factors that govern tissue tropism. Receptor engagement plays an essential role in viral replication and thus influences the course and outcome of viral disease. To understand the host range, tissue tropism, and virulence of this pathogen, it is critical to elucidate molecular mechanisms of CHIKV attachment and cell entry. Identifying the attachment molecules to which CHIKV binds is a critical step in obtaining a more comprehensive analysis of the envelope-receptor interactions in CHIKV pathobiology. In addition, understanding key determinants of receptor utilization may provide insight into the factors that facilitate transmission between vector and host. This application investigates the viral and cellular determinants of CHIKV cell entry and will further an understanding of mechanisms by which CHIKV enters host cells. In Specific Aim 1, we will determine the role of N-linked glycosylation sites in the viral envelope proteins in viral replication. Wild-type and glycosylation-site mutant viruses will be produced in mammalian and mosquito cells. The utilization and type of glycosylation at each site will be determined. Cell binding, infectivity, and replication of mutant viruses will be assessed. In Specific Aim 2, the role of cell-surface glycans in CHIKV replication will be investigated. Types of glycans to which CHIKV binds will be identified by glycan array, and their role in virus binding and cell entry will be analyzed. In Specific Aim 3, host proteins that mediate viral entry will be identified using proteomic and genomic strategies. Candidate receptors will be analyzed for their role in facilitating virus infection. These studies will provide valuable insights into mechanisms of CHIKV cell entry and further elucidate virus-host interactions at a critical stage of viral replication. Moreover, findings from these studies will provide new insights into the tissue tropism and pathophysiology of CHIKV, which may illuminate new strategies for therapeutic intervention.